The long-term goals of this proposal are to understand how the alternative splicing of the Drosophila Down syndrome cell adhesion molecule (Dscam) gene is regulated and to determine the mechanism by which Dscam alternative splicing is mutually exclusive. The Dscam gene encodes an axon guidance receptor that plays an important role in neural development and is the most extensively alternatively spliced gene known to date. The Dscam gene contains 115 exons, 95 of which are alternatively spliced. The alternative exons are organized into 4 distinct clusters containing 12, 48, 33 and 2 mutually exclusive exons each. Because the exons within each cluster are alternatively spliced in a mutually exclusive manner, it is possible that 38,016 different Dscam isoforms can be expressed. It has been proposed that each Dscam isoform might interact with a different set of axon guidance cues and that the collection of Dscam isoforms expressed by a cell will be directly involved in guiding neurons to different addresses. It is therefore likely that individual neurons must in some way be programmed to splice the Dscam pre-mRNA in specific ways. Thus understanding the mechanisms regulating Dscam alternative splicing will provide insight into the genetic program that specifies neural wiring. This proposal is aimed at understanding the mechanisms involved in regulating the alternative splicing of the Dscam exon 4 cluster which contains 12 mutually exclusive exons. First, we will identify RNA sequences involved in the regulation of Dscam alternative splicing and the proteins that bind to these elements. Second, we will determine the mechanism involved in the developmental regulation of exon 4.2 alternative splicing. Third, we will determine the mechanism by which the SR protein B52 and the general splicing factor dU2AF modulate exon 4.4 alternative splicing. Finally, we will determine the mechanistic basis by which alternative splicing of the Dscam exon 4 cluster is mutually exclusive. Together, these experiments will provide significant insight into the mechanisms involved in regulating alternative splicing, the mechanism responsible for mutually exclusive alternative splicing, and the genetic program that determines the specificity of neural wiring in Drosophila. [unreadable] [unreadable]